The present invention relates generally to synthetic analogues of the polypeptide hormone .alpha.-melanotropin and more particularly to cyclic analogues which exhibit increased potency, prolongation and serum stability characteristics in comparison to the linear native hormone.
.alpha.-Melanotropin (.alpha.-MSH, .alpha.-melanocyte stimulating hormone) is a linear tridecapeptide synthesized and secreted by the pars intermedia of the vertebrate pituitary. By convention, the amino acid residues of this linear tridecapeptide are numbered sequentially from the amino terminal (here acetyl substituted) carbon atom through the carboxy terminal (here carboxamide terminal) as follows: EQU Ac-Ser.sup.1 -Tyr.sup.2 -Ser.sup.3 -Met.sup.4 -Glu.sup.5 -His.sup.6 -Phe.sup.7 -Arg.sup.8 -Trp.sup.9 -Gly.sup.10 -Lys.sup.11 -Pro.sup.12 -Val.sup.13 -NH.sub.2.
In this formula the following abbreviations are used: Ser=serine; Tyr=tyrosine; Met=methionine; Glu=glutamic acid; His=histidine; Phe=phenylalanine; Arg=arginine; Trp=tryptophan; Gly=glycine; Lys=lysine; Pro=proline; Val=valine.
.alpha.-MSH reversibly darkens amphibian skins by stimulating melanosome movement (dispersion) within melanophores. .alpha.-Melanotropin also affects both normal and transformed (melanoma) mammalian melanocytes by stimulating adenylate cyclase activity, tyrosinase activity and melanin production. In addition, recent studies suggest that the native hormone may have important physiological roles in the control of vertebrate pigment cell melanogenesis (See, Hadley, et al., Peptides of the Pars Intermedia, 81 (Evered, et al., Eds.), 242-261, Pitman Medical, London.)) and important functions in fetal development and in neural mechanisms related to learning and memory. See, Front. Horm. Res., 4, (Tilders, et al., Eds.), S. Karger, Basil (1977).
The recognition that .alpha.-MSH functions in a number of roles in mammals, including humans, in addition to its well-characterized role in the color change mechanism of poikilothermic vertebrates and its effect on melanoma cell activity and growth, has prompted substantial research into production and testing of synthetic .alpha.-MSH and analogues thereof.
Variation in biological effects of some .alpha.-MSH analogues generally can be discussed in terms of "potentiation" (increased activity relative to naturally-occurring .alpha.-MSH) and "prolongation". Such effects are measured by the classic frog skin bioassay system (See, Shizume, et al., Endocrinology, 54, 553-560 (1954)), or the lizard skin bioassay system (See, Hadley, et al., Science, 213, 1025-1027 (1981); Huntington, et al., Endocrinology, 66, 599-609 (1970)). Because .alpha.-MSH has an extremely short half-life in serum (less than 2 minutes), synthetic analogues having greater serum stability in addition to greater potency and prolongation characteristics have been sought.
Of particular interest to the background of the invention are studies of the structure-function relationships of .alpha.-MSH through analysis of the biological activity of a number of structurally or stereochemically modified .alpha.-MSH analogues. The amino acid residue sequences of .alpha.-MSH believed to be important for expression of melanotropic activity (the so-called "active sites") have been identified. It has been suggested that .alpha.-MSH contains two active sites, (Glu.sup.5)-His.sup.6 -Pre.sup.7 -Arg.sup.8 -Trp.sup.9 and Gly.sup.10 -Lys.sup.11 -Pro.sup.12 -Val.sup.13 -NH.sub.2. See, Eberle, Cellular Receptors for Hormones and Neurotransmitters (Schluster, et al., Eds.), New York (1980), pp. 219-231. Each of these sequences of .alpha.-MSH can apparently stimulate melanosome dispersion in amphibian melanophores in vitro. See, Schwyzer, Ann. N.Y. Acad. Sci., 217, 3-26 (1977). Studies of the biological activity of stereochemically modified .alpha.-MSH analogues of .alpha.-MSH have also provided important insights relative to the structural requirements of the native hormone for receptor binding and subsequent melanotropic expression. Substitution of the D-isomeric configuration of the amino acid residue phenylalanine at position 7 and the amino acid residue norleucine at position 4 of the native hormone has led to an .alpha.-MSH analogue [Nle.sup.4, D-Phe.sup.7 ]-.alpha.-MSH which exhibits significant increases in potency, prolongation and greater stability toward enzymatic degradation. See, Sawyer, et al., P.N.A.S., 77, 5754-5758 (1980). Specifically incorporated by reference to illustrate the background of the invention and the prior art is the inventors' co-pending patent application Ser. No. 341,387, filed Oct. 23, 1981.
Also of interest to the background of the present invention are studies which suggest that .alpha.-MSH is a conformationally flexible linear molecule in aqueous solution. See, Schwyzer, supra. However, experimental research into the biologically active conformation of .alpha.-MSH, i.e., the three-dimensional configuration of the native hormone at the moment it is bound to its receptor and elicits signal transduction resulting in a biological response, has not been elucidated.
There continues to exist, therefore, a long-standing need in the art for physiologically stable, highly potent analogues of .alpha.-MSH which exhibit prolonged biological response and resistance to enzymatic degradation.
Specifically incorporated by reference herein for purposes of illustrating the background of the present invention and the prior art is the publication by the inventors and their co-workers appearing in P.N.A.S., 79, pp. 1751-1755 (March, 1982).